Methods of making L-amino acids in coryneform bacteria using the sigE gene

ABSTRACT

The present invention relates to an isolated polynucleotide from  Corynebacterium glutamicum  comprising a polynucleotide sequence chosen from the group consisting of (a) a polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequence of SEQ ID NO: 2; (b) a polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequence of SEQ ID NO: 2; (c) a polynucleotide which is complementary to the polynucleotides of (a) or (b); and (d) a polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequence of (a), (b), or (c), and a process for the fermentative preparation of L-amino acids using coryneform bacteria in which at least the sigE gene is present in enhanced form, and the use of polynucleotides which comprise the sequence according to the invention as hybridization probes.

FIELD OF THE INVENTION

The invention provides nucleotide sequences from coryneform bacteriawhich code for the sigE gene and a process for the fermentativepreparation of amino acids using bacteria in which the sigE gene isenhanced.

PRIOR ART

L-Amino acids are used in human medicine and in the pharmaceuticalsindustry, in the foodstuffs industry and especially in animal nutrition.

It is known that amino acids are prepared by fermentation from strainsof coryneform bacteria, in particular Corynebacterium glutamicum.Because of their great importance, work is constantly being undertakento improve the preparation processes. Improvements to the process canrelate to fermentation measures, such as, for example, stirring andsupply of oxygen, or the composition of the nutrient media, such as, forexample, the sugar concentration during the fermentation, or the workingup to the product form by, for example, ion exchange chromatography, orthe intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and produce amino acids are obtained in thismanner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of Corynebacterium strains whichproduce L-amino acid, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

OBJECT OF THE INVENTION

The inventors had the object of providing new measures for improvedfermentative preparation of amino acids.

SUMMARY OF THE INVENTION

Where L-amino acids or amino acids are mentioned in the following, thismeans one or more amino acids, including their salts, chosen from thegroup consisting of L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan and L-arginine. Lysine is particularly preferred.

The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence which codes for the sigEgene, chosen from the group consisting of

-   -   a) polynucleotide which is identical to the extent of at least        70% to a polynucleotide which codes for a polypeptide which        comprises the amino acid sequence of SEQ ID No. 2,    -   b) polynucleotide which codes for a polypeptide which comprises        an amino acid sequence which is identical to the extent of at        least 70% to the amino acid sequence of SEQ ID No. 2,    -   c) polynucleotide which is complementary to the polynucleotides        of a) or b), and    -   d) polynucleotide comprising at least 15 successive nucleotides        of the polynucleotide sequence of a), b) or c),    -   the polypeptide preferably having the activity of sigma factor        E.

The invention also provides the above-mentioned polynucleotide, thispreferably being a DNA which is capable of replication, comprising:

-   -   (i) the nucleotide sequences shown in SEQ ID No. 1, SEQ ID NO. 3        or 4, or    -   (ii) at least one sequence which corresponds to sequence (i)        within the range of the degeneration of the genetic code, or    -   (iii) at least one sequence which hybridizes with the sequence        complementary to sequence (i) or (ii), and optionally    -   (iv) sense mutations of neutral function in (i).

The invention also provides

-   -   a polynucleotide, in particular DNA, which is capable of        replication and comprises the nucleotide sequence as shown in        SEQ ID No. 1;    -   a polynucleotide which codes for a polypeptide which comprises        the amino acid sequence as shown in SEQ ID No. 2;    -   a vector containing the polynucleotide according to the        invention, in particular a shuttle vector or plasmid vector, and    -   coryneform bacteria which contain the vector or in which the        sigE gene is enhanced.

The invention also provides polynucleotides which substantially comprisea polynucleotide sequence, which are obtainable by screening by means ofhybridization of a corresponding gene library of a coryneform bacterium,which comprises the complete gene or parts thereof, with a probe whichcomprises the sequence of the polynucleotide according to the inventionaccording to SEQ ID No. 1 or a fragment thereof, and isolation of thepolynucleotide sequence mentioned.

DETAILED DESCRIPTION OF THE INVENTION

Polynucleotides which comprise the sequences according to the inventionare suitable as hybridization probes for RNA, cDNA and DNA, in order toisolate, in the full length, nucleic acids or polynucleotides or geneswhich code for sigma factor E or to isolate those nucleic acids orpolynucleotides or genes which have a high similarity of sequence withthat of the sigE gene.

Polynucleotides which comprise the sequences according to the inventionare furthermore suitable as primers with the aid of which DNA of geneswhich code for sigma factor E can be prepared by the polymerase chainreaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least30, preferably at least 20, very particularly preferably at least 15successive nucleotides. Oligonucleotides which have a length of at least40 or 50 nucleotides are also suitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention include a polynucleotideaccording to SEQ ID No. 1 or a fragment prepared therefrom and alsothose which are at least 70%, preferably at least 80% and in particularat least 90% to 95% identical to the polynucleotide according to SEQ IDNo. 1 or a fragment prepared therefrom.

“Polypeptides” are understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of sigma factor E, and also those which are at least 70%,preferably at least 80% and in particular at least 90% to 95% identicalto the polypeptide according to SEQ ID No. 2 and have the activitymentioned.

The invention furthermore relates to a process for the fermentativepreparation of amino acids chosen from the group consisting ofL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginineusing coryneform bacteria which in particular already produce aminoacids and in which the nucleotide sequences which code for the sigE geneare enhanced, in particular over-expressed.

The term “enhancement” in this connection describes the increase in theintracellular activity of one or more enzymes in a microorganism whichare coded by the corresponding DNA, for example by increasing the numberof copies of the gene or allele or of the genes or alleles, using apotent promoter or using a gene or allele which codes for acorresponding enzyme having a high activity, and optionally combiningthese measures.

The microorganisms which the present invention provides can produceL-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), are in particularthe known wild-type strains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Corynebacterium melassecola ATCC17965.    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        and L-amino acid-producing mutants or strains prepared        therefrom.

The new sigE gene from C. glutamicum which codes for the enzyme sigmafactor E has been isolated.

To isolate the sigE gene or also other genes of C. glutamicum, a genelibrary of this microorganism is first set up in Escherichia coli (E.coli). The setting up of gene libraries is described in generally knowntextbooks and handbooks. The textbook by Winnacker: Gene und Klone, EineEinführung in die Gentechnologie (Verlag Chemie, Weinheim, Germany,1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50,495-508 (1987)). Bathe et al. (Molecular and General Genetics,252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032,which was set up with the aid of the cosmid vector SuperCos I (Wahl etal., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575).

Börmann et al. (Molecular Microbiology 6 (3), 317-326) (1992)) in turndescribe a gene library of C. glutamicum ATCC13032 using the cosmidpHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).

To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25,807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268).Suitable hosts are, in particular, those E. coli strains which arerestriction- and recombination-defective. An example of these is thestrain DH5αmcr, which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNAfragments cloned with the aid of cosmids can in turn be subcloned in theusual vectors suitable for sequencing and then sequenced, as isdescribed e.g. by Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232 (1986)), that of Marck (NucleicAcids Research 16, 1829-1836 (1988)) or the GCG program of Butler(Methods of Biochemical Analysis 39, 74-97 (1998)).

The new DNA sequence of C. glutamicum which codes for the sigE gene andwhich, as SEQ ID No. 1, is a constituent of the present invention hasbeen found. The amino acid sequence of the corresponding protein hasfurthermore been derived from the present DNA sequence by the methodsdescribed above. The resulting amino acid sequence of the sigE geneproduct is shown in SEQ ID No. 2.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy ofthe genetic code are also a constituent of the invention. In the sameway, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ IDNo. 1 are a constituent of the invention. Conservative amino acidexchanges, such as e.g. exchange of glycine for alanine or of asparticacid for glutamic acid in proteins, are furthermore known among expertsas “sense mutations” which do not lead to a fundamental change in theactivity of the protein, i.e. are of neutral function. It is furthermoreknown that changes on the N and/or C terminus of a protein cannotsubstantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID No. 2 are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found by the expert, inter alia, in the handbook “The DIG SystemUsers Guide for Filter Hybridization” from Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology (1991) 41: 255-260). The hybridization takesplace under stringent conditions, that is to say only hybrids in whichthe probe and target sequence, i.e. the polynucleotides treated with theprobe, are at least 70% identical are formed. It is known that thestringency of the hybridization, including the washing steps, isinfluenced or determined by varying the buffer composition, thetemperature and the salt concentration. The hybridization reaction ispreferably carried out under a relatively low stringency compared withthe washing steps (Hybaid Hybridisation Guide, Hybaid Limited,Teddington, UK, 1996).

A 5×SSC buffer at a temperature of approx. 50-68° C., for example, canbe employed for the hybridization reaction. Probes can also hybridizehere with polynucleotides which are less than 70% identical to thesequence of the probe. Such hybrids are less stable and are removed bywashing under stringent conditions. This can be achieved, for example,by lowering the salt concentration to 2×SSC and optionally subsequently0.5×SSC (The DIG System User's Guide for Filter Hybridisation,Boehringer Mannheim, Mannheim, Germany, 1995) a temperature of approx.50-68° C. being established. It is optionally possible to lower the saltconcentration to 0.1×SSC. Polynucleotide fragments which are, forexample, at least 70% or at least 80% or at least 90% to 95% identicalto the sequence of the probe employed can be isolated by increasing thehybridization temperature stepwise from 50 to 68° C. in steps of approx.1-2° C. Further instructions on hybridization are obtainable on themarket in the form of so-called kits (e.g. DIG Easy Hyb from RocheDiagnostics GmbH, Mannheim, Germany, Catalogue No. 1603558).

Instructions for amplification of DNA sequences with the aid of thepolymerase chain reaction (PCR) can be found by the expert, inter alia,in the handbook by Gait: Oligonucleotide Synthesis: A Practical Approach(IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994).

In the work on the present invention, it has been found that coryneformbacteria produce amino acids in an improved manner after over-expressionof the sigE gene.

To achieve an over-expression, the number of copies of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same way. By inducible promoters, it is additionallypossible to increase the expression in the course of fermentative aminoacid production. The expression is likewise improved by measures toprolong the life of the m-RNA. Furthermore, the enzyme activity is alsoincreased by preventing the degradation of the enzyme protein. The genesor gene constructs can either be present in plasmids with a varyingnumber of copies, or can be integrated and amplified in the chromosome.Alternatively, an over-expression of the genes in question canfurthermore be achieved by changing the composition of the media and theculture procedure.

Instructions in this context can be found by the expert, inter alia, inMartin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP 0472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler(Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied andEnvironmental Microbiology 60, 126-132 (1994)), in LaBarre et al.(Journal of Bacteriology 175, 1001-1007 (1993)), in WO 96/15246, inMalumbres et al. (Gene 134, 15-24 (1993)), in JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks of genetics and molecular biology.

By way of example, for enhancement the sigE gene according to theinvention was over-expressed with the aid of episomal plasmids. Suitableplasmids are those which are replicated in coryneform bacteria. Numerousknown plasmid vectors, such as e.g. pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al.,Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991))are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmidvectors, such as e.g. those based on pCG4 (U.S. Pat. No. 4,489,160), orpNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124(1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in the samemanner.

Plasmid vectors which are furthermore suitable are also those with theaid of which the process of gene amplification by integration into thechromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO(Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat.No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernard etal., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpfet al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt etal., 1986, Gene 41: 337-342). The plasmid vector which contains the geneto be amplified is then transferred into the desired strain of C.glutamicum by conjugation or transformation. The method of conjugationis described, for example, by Schäfer et al. (Applied and EnvironmentalMicrobiology 60, 756-759 (1994)). Methods for transformation aredescribed, for example, by Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123,343-347 (1994)). After homologous recombination by means of a “crossover” event, the resulting strain contains at least two copies of thegene in question.

In addition, it may be advantageous for the production of L-amino acidsto enhance, in particular over-express one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the sigE gene.

Thus, for example, for the preparation of L-amino acids, in addition toenhancement of the sigE gene, one or more genes chosen from the groupconsisting of

-   -   the dapA gene which codes for dihydrodipicolinate synthase (EP-B        0 197 335),    -   the gap gene which codes for glyceraldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086),    -   the tpi gene which codes for triose phosphate isomerase        (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),    -   the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the zwf gene which codes for glucose 6-phosphate dehydrogenase        (JP-A-09224661),    -   the pyc gene which codes for pyruvate carboxylase (DE-A-198 31        609),    -   the mqo gene which codes for malate-quinone oxidoreductase        (Molenaar et al., European Journal of Biochemistry 254, 395-403        (1998)),    -   the lysC gene which codes for a feed-back resistant aspartate        kinase (Accession No. P26512; EP-A-0699759),    -   the lysE gene which codes for lysine export (DE-A-195 48 222),    -   the hom gene which codes for homoserine dehydrogenase (EP-A        0131171),    -   the ilvA gene which codes for threonine dehydratase (Möckel et        al., Journal of Bacteriology (1992) 8065-8072)) or the ilvA(Fbr)        allele which codes for a “feed back resistant” threonine        dehydratase (Möckel et al., (1994) Molecular Microbiology 13:        833-842),    -   the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B        0356739),    -   the ilvD gene which codes for dihydroxy-acid dehydratase (Sahm        and Eggeling (1999) Applied and Environmental Microbiology 65:        1973-1979),    -   the zwa1 gene which codes for the Zwa1 protein (DE: 19959328.0,        DSM 13115)        can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of L-amino acids,in addition to the enhancement of the sigE gene, for one or more of thegenes chosen from the group consisting of:

-   -   the pck gene which codes for phosphoenol pyruvate carboxykinase        (DE 199 50 409.1; DSM 13047),    -   the pgi gene which codes for glucose 6-phosphate isomerase (U.S.        Ser. No. 09/396,478; DSM 12969),    -   the poxB gene which codes for pyruvate oxidase (DE: 1995 1975.7;        DSM 13114),    -   the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2,        DSM 13113)        to be attenuated, in particular for the expression thereof to be        reduced.

In addition to over-expression of the sigE gene it may furthermore beadvantageous for the production of amino acids to eliminate undesirableside reactions (Nakayama: “Breeding of Amino Acid ProducingMicro-organisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The invention also provides the microorganisms prepared according to theinvention, and these can be cultured continuously or discontinuously inthe batch process (batch culture) or in the fed batch (feed process) orrepeated fed batch process (repetitive feed process) for the purpose ofproduction of amino acids. A summary of known culture methods isdescribed in the textbook by Chmiel (Bioprozesstechnik 1. Einführung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fattyacids, such as e.g. palmitic acid, stearic acid and linoleic acid,alcohols, such as e.g. glycerol and ethanol, and organic acids, such ase.g. acetic acid, can be used as the source of carbon. These substancescan be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as e.g. magnesium sulfate or iron sulfate, whichare necessary for growth. Finally, essential growth substances, such asamino acids and vitamins, can be employed in addition to theabove-mentioned substances. Suitable precursors can moreover be added tothe culture medium. The starting substances mentioned can be added tothe culture in the form of a single batch, or can be fed in during theculture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as e.g. fatty acid polyglycol esters, canbe employed to control the development of foam. Suitable substanceshaving a selective action, such as e.g. antibiotics, can be added to themedium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e.g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of the desired product has formed. This targetis usually reached within 10 hours to 160 hours.

Methods for the determination of L-amino acids are known from the priorart. The analysis can thus be carried out, for example, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) by ion exchangechromatography with subsequent ninhydrin derivation, or it can becarried out by reversed phase HPLC, for example as described by Lindrothet al. (Analytical Chemistry (1979) 51: 1167-1174).

The process according to the invention is used for fermentativepreparation of amino acids.

The following microorganism was deposited as a pure culture on 11th Apr.2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ=German Collection of Microorganisms and Cell Cultures,Braunschweig, Germany) in accordance with the Budapest Treaty:

-   -   Corynebacterium glutamicum DSM5715/pEC-T18mob2sigEexp as DSM        14229.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques ofrestriction, Klenow and alkaline phosphatase treatment were carried outby the method of Sambrook et al. (Molecular Cloning. A Laboratory Manual(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,USA). Methods for transformation of Escherichia coli are also describedin this handbook.

The composition of the usual nutrient media, such as LB or TY medium,can also be found in the handbook by Sambrook et al.

EXAMPLE 1

Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolatedas described by Tauch et al. (1995, Plasmid 33:168-179) and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP,Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al.(1987) Proceedings of the National Academy of Sciences USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner was mixed withthe treated ATCC13032 DNA and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no. 27-0870-04). The ligation mixture was thenpacked in phages with the aid of Gigapack II XL Packing Extract(Stratagene, La Jolla, USA, Product Description Gigapack II XL PackingExtract, Code no. 200217).

For infection of the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcid Research 16:1563-1575) the cells were taken up in 10 mM MgSO₄ andmixed with an aliquot of the phage suspension. The infection andtitering of the cosmid library were carried out as described by Sambrooket al. (1989, Molecular Cloning: A laboratory Manual, Cold SpringHarbor), the cells being plated out on LB agar (Lennox, 1955, Virology,1:190) with 100 mg/l ampicillin. After incubation overnight at 37° C.,recombinant individual clones were selected.

EXAMPLE 2

Isolation and Sequencing of the sigE Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partly cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (RocheDiagnostics GmbH, Mannheim, Germany, Product Description SAP, ProductNo. 1758250). After separation by gel electrophoresis, the cosmidfragments in the size range of 1500 to 2000 bp were isolated with theQiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1, obtained from Invitrogen(Groningen, Holland, Product Description Zero Background Cloning Kit,Product No. K2500-01), was cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Product No. 27-0868-04). The ligation of the cosmid fragments in thesequencing vector pZero-1 was carried out as described by Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

The plasmid preparation of the recombinant clones was carried out withthe Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing was carried out by the dideoxy chain termination method ofSanger et al. (1977, Proceedings of the National Academy of SciencesU.S.A., 74:5463-5467) with modifications according to Zimmermann et al.(1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin TerminatorCycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction were carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0.The individual sequences of the pZero1 derivatives were assembled to acontinuous contig. The computer-assisted coding region analysis wasprepared with the XNIP program (Staden, 1986, Nucleic Acids Research,14:217-231).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis ofthe nucleotide sequence showed an open reading frame of 651 base pairs,which was called the sigE gene. The sigE gene codes for a protein of 216amino acids (SEQ ID NO. 2).

The DNA sections lying upstream and downstream of SEQ ID NO. 1, whichare shown in SEQ ID NO. 3 and SEQ ID NO. 4, were identified in the samemanner. The sigE gene region extended by SEQ ID NO. 3 and SEQ ID NO. 4is shown in SEQ ID NO. 5.

EXAMPLE 3

Preparation of a Shuttle Vector pEC-T18mob2sigEexp for Enhancement ofthe sigE Gene in C. glutamicum

3.1. Cloning of the sigE Gene

From the strain ATCC 13032, chromosomal DNA was isolated by the methodof Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis ofthe sequence of the sigE gene known for C. glutamicum from example 2,the following oligonucleotides were chosen for the polymerase chainreaction (see SEQ ID No. 7 and SEQ ID No. 8). sigE1: 5′TAG TCA CCA CGGTTA AGC CT 3′ sigE2: 5′GCC TTG GTT CTT ACG AAC TG 3′

The primers shown were synthesized by ARK Scientific GmbH Biosystems(Darmstadt, Germany) and the PCR reaction was carried out by thestandard PCR method of Innis et al. (PCR protocols. A guide to methodsand applications, 1990, Academic Press) with Taq-Polymerase from Qiagen(Hilden, Germany). With the aid of the polymerase chain reaction, theprimers allow amplification of a DNA fragment approx. 2.03 kb in size,which carries the sigE gene.

The amplified DNA fragment of approx. 2.03 kb in size which carries thesigE gene was ligated with the TOPO TA Cloning® Kit from InvitrogenCorporation (Carlsbad, Calif., USA) in the vector pCR®2.1TOPO (Bernardet al., Journal of Molecular Biology, 234: 534-541 (1993)). The E. colistrain Top10 (Grant et al., Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649) was then transformed with theligation batch in accordance with the instructions of the manufacturerof the kit (Invitrogen Corporation, Carlsbad, Calif., USA). Selection ofplasmid-carrying cells was carried out by plating out the transformationbatch on LB Agar (Sambrook et al., Molecular cloning: a laboratorymanual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989), which had been supplemented with 50 mg/l kanamycin.Plasmid DNA was isolated from a transformant with the aid of the QIAprepSpin Miniprep Kit from Qiagen (Hilden, Germany) and checked by treatmentwith the restriction enzyme SphI and EcoRI with subsequent agarose gelelectrophoresis (0.8%). The DNA sequence of the amplified DNA fragmentwas checked by sequencing. The plasmid was called pCR2.1sigEexp. Thestrain was called E. coli Top10/pCR2.1sigEexp.

3.2. Preparation of the E. coli-C. glutamicum Shuttle Vector pEC-T18mob2

The E. coli-C. glutamicum shuttle vector was constructed according tothe prior art. The vector contains the replication region reg of theplasmid pGA1 including the replication effector per (U.S. Pat. No.5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532(1997)), the tetracycline resistance-imparting tetA(Z) gene of theplasmid pAG1 (U.S. Pat. No. 5,158,891; gene library entry at theNational Center for Biotechnology Information (NCBI, Bethesda, Md., USA)with the accession number AF121000), the replication region oriV of theplasmid pMB1 (Sutcliffe, Cold Spring Harbor Symposium on QuantitativeBiology 43, 77-90 (1979)), the lacZα gene fragment including the lacpromoter and a multiple cloning site (mcs) (Norrander, J. M. et al. Gene26, 101-106 (1983)) and the mob region of the plasmid RP4 (Simon et al.,(1983) Bio/Technology 1:784-791). The vector constructed was transformedin the E. coli strain DH5α (Hanahan, In: DNA cloning. A PracticalApproach. Vol. I. IRL-Press, Oxford, Washington D.C., USA).

Selection for plasmid-carrying cells was made by plating out thetransformation batch on LB agar (Sambrook et al., Molecular cloning: ALaboratory Manual. 2^(nd) Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), which had been supplemented with 5 mg/ltetracycline. Plasmid DNA was isolated from a transformant with the aidof the QIAprep Spin Miniprep Kit from Qiagen and checked by restrictionwith the restriction enzymes EcoRI and HindIII and subsequent agarosegel electrophoresis (0.8%). The plasmid was called pEC-T18mob2 and isshown in FIG. 1.

3.3. Cloning of sigE in the E. coli-C. glutamicum Shuttle VectorpEC-T18mob2

The E. coli-C. glutamicum shuttle vector pEC-T18mob2 described inexample 3.2 was used as the vector. DNA of this plasmid was cleavedcompletely with the restriction enzymes BamHI and SalI and thendephosphorylated with shrimp alkaline phosphatase (Roche DiagnosticsGmbH, Mannheim, Germany, Product Description SAP, Product No. 1758250).

The sigE gene was isolated from the plasmid pCR2.1sigEexp described inexample 3.1. by complete cleavage with the enzymes BamHI and SalI. ThesigE fragment 1930 bp in size was isolated from the agarose gel with theQiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The sigE fragment obtained in this manner was mixed with the preparedvector pEC-T18mob2 and the batch was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no. 27-0870-04). The ligation batch was transformedin the E. coli strain DH5αMCR (Hanahan, In: DNA cloning. A PracticalApproach. Vol. I. IRL-Press, Oxford, Washington D.C., USA). Selection ofplasmid-carrying cells was made by plating out the transformation batchon LB agar (Lennox, 1955, Virology, 1:190) with 5 mg/l tetracycline.After incubation overnight at 37° C., recombinant individual clones wereselected. Plasmid DNA was isolated from a transformant with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and cleaved with therestriction enzymes BamHI and SalI to check the plasmid by subsequentagarose gel electrophoresis. The plasmid obtained was calledpEC-T18mob2sigEexp. It is shown in FIG. 2.

EXAMPLE 4

Transformation of the Strain DSM5715 with the Plasmid pEC-T18mob2sigEexp

The strain DSM5715 was transformed with the plasmid pEC-T18mob2sigEexpusing the electroporation method described by Liebl et al., (FEMSMicrobiology Letters, 53:299-303 (1989)). Selection of the transformantstook place on LBHIS agar comprising 18.5 g/l brain-heart infusion broth,0.5 M sorbitol, 5 g/l Bacto-tryptone, 2.5 g/l Bacto-yeast extract, 5 g/lNaCl and 18 g/l Bacto-agar, which had been supplemented with 5 mg/ltetracycline. Incubation was carried out for 2 days at 33° C.

Plasmid DNA was isolated from a transformant by conventional methods(Peters-Wendisch et al., 1998, Microbiology, 144, 915-927), cleaved withthe restriction endonucleases BamHI and SalI, and the plasmid waschecked by subsequent agarose gel electrophoresis. The strain obtainedwas called DSM5715/pEC-T18mob2sigEexp.

EXAMPLE 5

Preparation of Lysine

The C. glutamicum strain DSM5715/pEC-T18mob2sigEexp obtained in example4 was cultured in a nutrient medium suitable for the production oflysine and the lysine content in the culture supernatant was determined.

For this, the strain was first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with tetracycline (5 mg/l))for 24 hours at 33° C. Starting from this agar plate culture, apre-culture was seeded (10 ml medium in a 100 ml conical flask). Thecomplete medium CgIII was used as the medium for the pre-culture.

Medium Cg III

NaCl 2.5 g/l Bacto-Peptone  10 g/l Bacto-Yeast extract  10 g/l Glucose(autoclaved separately)  2% (w/v)

The pH was brought to pH 7.4

Tetracycline (5 mg/l) was added to this. The pre-culture was incubatedfor 16 hours at 33° C. at 240 rpm on a shaking machine. A main culturewas seeded from this pre-culture such that the initial OD (660 nm) ofthe main culture was 0.05. Medium MM was used for the main culture.

Medium MM

CSL (corn steep liquor)   5 g/l MOPS (morpholinopropanesulfonic acid) 20 g/l Glucose (autoclaved separately)  50 g/l (NH₄)₂SO₄  25 g/l KH₂PO₄0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O  10 mg/l FeSO₄ * 7 H₂O  10mg/l MnSO₄ * H₂O 5.0 mg/l Biotin (sterile-filtered) 0.3 mg/l Thiamine *HCl (sterile-filtered) 0.2 mg/l L-Leucine (sterile-filtered) 0.1 g/lCaCO₃  25 g/l

The CSL, MOPS and the salt solution were brought to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions werethen added, as well as the CaCO₃ autoclaved in the dry state.

Culturing is carried out in a 10 ml volume in a 100 ml conical flaskwith baffles. Tetracycline (5 mg/l) was added. Culturing was carried outat 33° C. and 80% atmospheric humidity.

After 48 hours, the OD was determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount oflysine formed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivation with ninhydrin detection.

The result of the experiment is shown in Table 1. TABLE 1 OD Lysine HClStrain (660 nm) g/l DSM5715/pEC-T18mob2 12.2 13.14 DSM5715/pEC- 13.0714.09 T18mob2sigEexp

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Map of the plasmid pEC-T18mob2.

FIG. 2: Map of the plasmid pEC-T18mob2sigEexp.

The abbreviations and designations used have the following meaning.

-   -   per: Gene for controlling the number of copies from PGA1    -   oriV: ColE1-similar origin from pMB1    -   rep: Plasmid-coded replication region from C. glutamicum plasmid        pGA1    -   RP4mob: RP4 mobilization site    -   lacZ-alpha: lacZ gene fragment from E. coli    -   Tet: Resistance gene for tetracycline    -   sigE: sigE gene of C. glutamicum    -   BamHI: Cleavage site of the restriction enzyme BamHI    -   SalI: Cleavage site of the restriction enzyme SalI    -   sigE: sigE gene of C. glutamicum    -   BamHI: Cleavage site of the restriction enzyme BamHI    -   SalI: Cleavage site of the restriction enzyme SalI

1-20. (canceled)
 21. An isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence as set forth in SEQ ID NO: 1; (b) a nucleotide sequenceencoding the polypeptide as set forth in SEQ ID NO: 2; and (c) anucleotide sequence fully complementary to (a) or (b).
 22. A vectorcomprising the nucleic acid of claim
 21. 23. A bacterium comprising thevector of claim 22, wherein said bacterium is an E. coli or coryneformbacterium.
 24. A recombinant Corynebacterium glutamicum comprising anoverexpressed polynucleotide encoding a polypeptide having the aminoacid sequence of SEQ ID NO: 2, wherein overexpression is achieved byincreasing the copy number of said polynucleotide or by operably linkingpromoter to said polynucleotide.
 25. An isolated nucleic acid thatencodes a polypeptide comprising an amino acid sequence that is at least95% identical to the amino acid sequence of SEQ ID NO: 2, wherein saidpolypeptide has a biological activity of a sigma factor E protein.
 26. Avector comprising the nucleic acid of claim
 25. 27. A host cellcomprising the vector of claim
 26. 28. An isolated polynucleotide primeror probe of a nucleic acid fragment, wherein said fragment consists ofat least 30 consecutive nucleotides from SEQ ID NO: 1 or the fullcomplement of said fragment.
 29. An isolated primer or probe consistingof a nucleic acid fragment, wherein said fragment consists of at least40 consecutive nucleotides from SEQ ID NO: 1 or the full complement ofsaid fragment.
 30. The shuttle vector pEC-T 18mob2sigEexp having (a) a1930 bp nucleic acid fragment of SEQ ID NO: 4 which harbors the C.glutamicum gene; and (b) a restriction map as set forth in FIG.
 2. 31.The shuttle vector of claim 30, wherein the vector has been deposited inCorynebacterium glutamicum strain DSM5715/pEC-T18mob2sigEexp underaccession no. DSM
 14229. 32. An isolated polynucleotide comprisingnucleotides 236 to 907 of SEQ ID NO: 1.